Human Babesiosis in Taiwan: Asymptomatic Infection with a Babesia microti-like Organism in a Taiwanese Woman

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1997, p Vol. 35, No /97/$ Copyright 1997, American Society for Microbiology Human Babesiosis in Taiwan: Asymptomatic Infection with a Babesia microti-like Organism in a Taiwanese Woman CHIEN-MING SHIH, 1 * LI-PING LIU, 1 WEN-CHING CHUNG, 2 S. J. ONG, 3 AND CHIH-CHIEN WANG 4 Department of Parasitology and Tropical Medicine, National Defense Medical Center, 1 Department of Parasitology, Taipei Medical College, 2 Division of Malariology and Parasitology, National Institute of Preventive Medicine, 3 and Department of Pediatrics, Tri-Service General Hospital, 4 Taipei, Taiwan, Republic of China Received 7 May 1996/Returned for modification 3 July 1996/Accepted 21 October 1996 An asymptomatic Babesia infection was confirmed by laboratory diagnoses. The intraerythrocytic protozoan (designed TW1) isolated from a 51-year-old Taiwanese woman appeared to be morphologically consistent with small-form piroplasm, and measurements indicated that it had a body size of 1.5 to 2.5 m in diameter. The typical features of ring, binary, and tetrad forms were observed in Giemsa-stained thin blood smears. A persistent and low-grade parasitemia was established after hamster inoculation. Indirect immunofluorescentantibody reactivities indicate that this strain (TW1) of Babesia was serologically related to, but not identical to, the Babesia species (B. microti) that infects rodents. Antibody titers in the patient s sera combined with the clinical symptoms suggested that the present case was a chronic and subclinical babesial infection. A neighborhood human serologic survey indicated that the infection may have been acquired accidentally from an infected rodent and localized within the same family. Indeed, rodents from areas around the neighborhood were trapped, and a high prevalence (83%) of babesial infection was observed. The possible vector responsible for the transmission remains to be identified. * Corresponding author. Mailing address: Department of Parasitology and Tropical Medicine, National Defense Medical Center, P.O. Box , Taipei, Taiwan, Republic of China. Phone: Fax: Babesiosis is a tick-transmitted protozoal infection caused by an intraerythrocytic malaria-like organism that infects a wide variety of wild and domestic animals (23, 40). Since the time that the first case of human babesiosis was documented in Yugoslavia (37), numerous cases of human babesial infections have been reported in Europe and the United States (1, 2, 14 16, 24, 34 36, 39). The European cases have been attributed to infection with Babesia of bovine origin, and Ixodes ricinus ticks are thought to be the vector responsible for the transmission of infections (16). However, Babesia microti, a species of rodent origin, has been recognized as the etiological agent of human babesiosis in the United States (10), and the Lyme disease vector ticks (Ixodes dammini) are incriminated as the same vector for the transmission of human babesial infections on various coastal islands and the mainland (38) in the northeastern United States. Symptoms of human babesiosis commence 1 to 4 weeks after a person is bitten by infected ticks and are characterized by a gradual onset of malaise, anorexia, fatigue, mild to moderate fever, drenching sweats, and myalgia (32, 34). Although splenectomized, immunocompromised, and elderly persons appear to be at greatest risk of acquiring babesiosis in the form of a clinical illness (5, 18, 25, 30, 31), asymptomatic and subclinical cases of babesial infection have been reported (13, 19, 26, 34). Because parasitemias in infected persons may be exceedingly sparse (34), laboratory diagnosis that depends solely on a demonstration of babesial organisms within erythrocytes by examining Giemsa-stained thin films may not be sufficient for detection of subclinical exposure. In addition, inoculation of the patient s blood into hamsters to amplify the undetectable parasitemia (2, 4, 9, 11, 17) and serologic testing for diagnosing chronic babesial infection and identifying the species of Babesia causing the infection (6, 7, 33) are required for a fully confirmed laboratory diagnosis. No laboratory-confirmed case of human babesiosis had been reported in Taiwan. Although serologic surveys for human babesiosis had been conducted on Taiwan (20), the prevalence of babesial infection is thought to be rare among Taiwanese aborigines. In a zoonotic survey, the prevalence of babesial (small piroplasm) infection is fairly high among rodents in Taiwan (41). Because of morphologic similarities and host range, the Babesia isolates from rodents in Taiwan were considered geographic variants of B. microti. Here we report details of the first laboratory-confirmed case of human babesiosis in Taiwan, and we also provide the results of serologic testing in an attempt to identify the species of this patient s Babesia isolate (designed TW1). In addition, the possible reservoir host was also investigated. MATERIALS AND METHODS Case history. A 51-year-old woman from a rural area (Min-Shung, Chia-i Hsien) in southwestern Taiwan presented to the local hospital in early March 1994 with the chief complaints of headache, malaise, fatigue, dull pain in the upper abdomen, and frequently mild fever with chill during the previous few months. At the local hospital she had been diagnosed as having and was treated for gallstones with tender hepatosplenomegaly, but her symptoms did not improve after hospitalization. She was transferred to the Medical Center in Taipei, was rehospitalized from 15 April to 18 May 1994, and was given routine hematologic examinations and supportive therapy. The patient had a temperature of 38.3 C, on average, a normal leukocyte count ( / l) and platelet count ( / l), and an abnormal erythrocyte count ( / l) and hematocrit (27.8%). She also had a decreased level of hemoglobin (8.9 g/dl) and was suspected of having hemolytic anemia. After routine examinations, she was sent home at her request. On 16 June, we received one tube of an EDTA-preserved blood specimen collected from the patient, and Giemsa-stained blood smears were made and examined by light microscopy with an oil objective. Intraerythrocytic malaria-like organisms were detected in about 1 of 200 erythrocytes by oil immersion microscopy. The patient had not traveled abroad during the previous 10 years, and the typical features of ring, binary, and tetrad (Maltese cross) forms of the parasites were noted and characterized a possible infection with Babesia organisms. A 10-day course of therapy with oral quinine (650 mg, three times daily) and intravenous clindamycin (300 mg, four times daily) was given, and her symptoms improved thereafter. The parasitemia of the patient was followed every month by examining blood smears, and failure of clearance of the parasites from the 450

2 VOL. 35, 1997 HUMAN BABESIOSIS IN TAIWAN 451 peripheral blood was observed. On 20 September, she was retreated with a combination of oral quinine (650 mg, three times daily) and azithromycin (500 mg, twice daily) for 10 days. After treatment, no intraerythrocytic parasites were detected on her blood smears in October 1994 or on blood smears in November 1994, February 1995, and May Inoculation of the patient s blood into animals and serologic testing for antibody to Babesia parasites were performed for confirmatory diagnosis. Isolation of parasites. For isolation of piroplasms, golden Syrian hamsters (Mesocricetus auratus; weight, 50 to 60 g) were purchased from the animal supply center of National Taiwan University and were given whole-blood specimens from the patient intraperitoneally by injection (a 1-ml inoculum per hamster) (4, 29). Giemsa-stained thin smears of blood from the inoculated hamsters were examined (at least 25 random fields per smear) weekly by oil immersion microscopy for 6 months. The percent parasitemia and the persistence of the babesial infection in the inoculated hamsters were recorded. Serologic tests. Serial serum specimens collected from the patient were tested in serial twofold dilutions (from 1:16 to 1:2,048) by indirect immunofluorescentantibody (IFA) assay for antibody reactivity to B. microti and the patient s isolate (isolate TW1). Hamster erythrocytes parasitized with intraerythrocytic forms of B. microti (courtesy of Sam R. Telford III, Department of Tropical Public Health, Harvard University) or TW1 piroplasms (propagated in hamster blood) were prepared as antigens in the 10-well glass IFA slides (22). The prepared antigen slides were sealed in boxes and stored at 20 C until testing. In general, serum specimens diluted with phosphate-buffered saline (PBS) were added to each well of antigen-coated IFA slides. The slides were placed in a humid chamber, incubated for 30 min at 37 C, washed three times in PBS, and then allowed to air dry. A total of 20 l of fluorescein isothiocyanate (FITC)-conjugated, affinity-purified goat anti-human immunoglobulin G (IgG; F0132; Sigma Co.) diluted in PBS with Evans blue (0.01%) was added to each well, and the slides were allowed to incubate for 30 min at 37 C. After three washes with PBS, the slides were allowed to air dry and were mounted with buffered glycerin, and coverslips were placed on the slides. For all slides, 50 random fields were examined by epifluorescence microscopy with an oil objective. In an attempt to identify the species of the patient s isolate (strain TW1), high titers of antisera from various Babesia-infected humans (courtesy of Sam R. Telford III, Harvard University) and animals (courtesy of Patricia A. Conrad, University of California at Davis) were tested for their reactivities against TW1 and rat isolates. FITC-conjugated, affinity-purified goat anti-human IgG (F0132; Sigma) and rabbit anti-dog IgG (Jackson ImmunoResearch Labs, West Grove, Pa.) were used as secondary antibodies for analysis. A titer of reactivity of at least 64 to the TW1 and rat isolates was considered positive reactivity. Neighborhood serologic survey. Whole-blood specimens from the patient s family members and persons who lived near the patient s home were collected in anticoagulant-coated blood collection tubes (Vacutainer 6457; Taiwan branch of Becton Dickinson) on 23 September After differential centrifugation, the serum specimens were obtained and assayed for their IFA assay reactivities to B. microti, the rat isolate, or the TW1 isolate as described previously (29). An antibody reactivity titer to B. microti, the rat isolate, or the TW1 isolate of at least 64 was considered seropositivity. The remaining whole-blood specimens from seropositive persons were inoculated into hamsters to amplify and identify the isolate causing the suspected babesial infection. Giemsa-stained thin smears of the inoculated hamsters were examined and followed for 8 weeks. Epizootiologic survey. Small mammals from various sites near the patient s home were trapped from September to December After anesthetization, whole-blood specimens were obtained by cardiac puncture from 35 rodents. These rodents included 8 black rats (Rattus rattus, Linnaeus), 12 spinous country rats (Rattus coxinga, Swinhoe), and 15 brown rats (Rattus norvegicus, Erxleben). Thin blood smears were made from each rodent s blood, and Giemsa-stained smears were examined for evidence of babesial infection by oil immersion microscopy. The remaining blood specimens were inoculated into hamsters to amplify the possible occult infection, and thin smears were examined weekly for 8 weeks. Attempts to collect live ticks during the trapping seasons were not successful. RESULTS The intraerythrocytic organism observed in the Giemsastained thin blood smears of the patient s blood was a smallform piroplasm measuring about 1.5 to 2.5 m in diameter. The typical feature of the parasite was a red dot of chromatin with a bit of cytoplasm (Fig. 1a). The predominant forms in most of the blood smears closely resembled the ring form of Plasmodium spp., with small to large cytoplasmic vacuoles. Two or more chromatin masses with large cytoplasmic vacuolated areas were seen in more mature forms of the parasite (Fig. 1b), and multiply infected erythrocytes with the tetrad form of parasite were occasionally seen in the smears (Fig. 1c). There was no significant enlargement of the erythrocytes, and no evidence of pigment deposition was noted in the infected erythrocytes. These features indicate that the patient was infected with a Babesia parasite. To characterize the course of parasitemia of the Babesia isolate (isolate TW1), hamsters intact with spleens were inoculated individually with a 1.0-ml specimen of whole blood from the patient, and thin smears of peripheral blood from the inoculated hamsters were examined for the presence of babesial infection. The features of ring, binary, and tetrad forms of the parasites were noted (Fig. 1d to f). The parasitemia was observed in about 1 of 200 erythrocytes (0.5%) during the first week after inoculation and gradually increased to the rate of 4.5% at 4 weeks after inoculation (data not shown). The infection rate declined to a scarce level ( 1%) at 8 weeks after inoculation and became undetectable 4 months thereafter. These observations indicate that the babesial isolate from the patient can persist in the inoculated hamsters with a low-grade parasitemia for several months. To determine the titer of antibody against the Babesia parasites in the patient s serum, serial serum specimens were collected and tested for IFA reactivity to B. microti and the TW1 isolate. Antibody titers of 512 to TW1 and 64 to B. microti were detected in the serum sample collected on 16 June The antibody reactivity dropped 2 weeks after treating the patient with quinine and clindamycin, as determined with a blood specimen on 18 July 1994, and titers of 128 to TW1 and 32 to B. microti were sustained for almost 3 months (Table 1). The reactivities declined dramatically at 2 weeks following retreatment with a combination of quinine and azithromycin, as determined with a blood specimen obtained on 5 October 1994, and no parasite was detected in the patient s blood smears thereafter. In addition, antibody titers waned to an undetectable level over the months after retreatment. These results suggest that this babesial infection may be attributed to a chronic and subclinical exposure. Furthermore, three species of rodents from areas around the patient s neighborhood were trapped and examined for the prevalence of Babesia infection by routine techniques of examining blood smears and inoculating blood into hamsters. Of the 35 rats (8 R. rattus, 12R. coxinga, and 15 R. norvegicus rats), only R. coxinga had evidence of babesial infection. The infection rate was shown to be about 67% (8 of 12 animals) and 83% (10 of 12 animals) as determined by examining blood smears and inoculating hamsters, respectively. Moreover, both the organism observed in the smears of rat blood and the isolates from rats detected in hamster blood were morphologically similar to the patient s isolate (isolate TW1). These results suggest that rodents at the sites where the disease was endemic may be the reservoir hosts responsible for the zoonotic transmission of the babesial infection. Seroreactivity was also done to determine whether the species of the patient s isolate (isolate TW1) could be identified by differential IFA reactivity with high titers of antisera from various Babesia-infected humans and animals. Antibody reactivity with the TW1 isolate was observed to be very strong (titer of 64 or higher) with antisera against B. microti, but only weak reactivity was observed with antiserum against B. gibsoni or B. canis (Table 2). In addition, there was very reactivity with the rat isolate with antiserum against B. microti, and very low reactivity was observed with antiserum against canine Babesia. These results indicate that the species of the patient s isolate (TW1) was serologically related to B. microti. To determine whether transmission of Babesia infection is prevalent near the patient s home and whether the infection is endemic, serum specimens were collected from 38 people (members of six families and 13 neighbors) and tested for IFA

3 452 SHIH ET AL. J. CLIN. MICROBIOL. FIG. 1. Photomicrographs of Giemsa-stained blood films from the patient (a to c) and inoculated hamster (d to f). (a) Ring-form trophozoite of a small Babesia organism observed in the patient s blood, consisting of a red dot of chromatin with a bit of cytoplasm. Magnification, 2,500. (b) Mature trophozoite stage with three chromatin dots and a large vacuolated area. Magnification, 2,500. (c) Multiply infected erythrocyte with tetrad form of parasite. Magnification, 2,500. (d) Ring-stage trophozoite with a chromatin mass and a wisp of blue cytoplasm observed in the blood of an inoculated hamster. Magnification, 1,250. (e) Binucleate trophozoite observed in the blood of an inoculated hamster. Magnification, 1,250. (f) Tetrad form of parasite observed in the blood of an inoculated hamster. Magnification, 1,800. reactivity to TW1, the rat isolate, and B. microti. Of the 38 people, 14 (36.8%) were female, with an age range of from 2 to 56 years. Only two serum samples, from female participants, showed seroreactivity with the TW1 and rat isolates, and antibody titers of 128 and 64 to the TW1 and the rat isolate, respectively, were observed (data not shown). These two seropositive persons included one family member of the patient and the patient. Both of them are housekeepers, and neither patient had a history of travel during the previous few years. The antibody reactivity to B. microti was noted only with the patient s serum, and hamster inoculation of whole blood from the family member suspected of being infected did not result in

4 VOL. 35, 1997 HUMAN BABESIOSIS IN TAIWAN 453 TABLE 1. IFA reactivity of serial serum specimens from the patient with rodent (B. microti) and human (TW1) babesial isolates Date of specimen collection Reciprocal antibody titer a to: B. microti TW1 isolate a detectable parasitemia. These results suggest that the babesial infection may have been acquired accidentally from an infected rodent and localized within the same family. DISCUSSION Parasites detected in blood smear 16 Jun Jul 1994 b Aug Sep Oct 1994 c Nov Feb May a B. microti- and TW1-infected erythrocytes of hamsters were prepared as antigens in a 10-well IFA assay slide. b Two weeks after routine treatment for babesial infection. c Two weeks after retreatment with oral quinine and azithromycin. Our report describes the first definitive case of human babesiosis acquired in Taiwan. Because of the high prevalence of hepatitis infections in Taiwan, the patient was initially misdiagnosed as having hepatosplenomegaly and had been treated with supportive therapy by the local hospital. The patient was also suspected of having hemolytic anemia due to the decreased level of hemoglobin combined with an abnormal hematocrit value. However, a suspicious organism detected within the erythrocytes led us to proceed with a careful screening for intracellular parasites, and organisms with features typical of those of Babesia parasites were observed in Giemsastained thin smears. The intraerythrocytic parasite was morphologically consistent with the rodent Babesia (B. microti). Malaria infection was ruled out not only by the evidence that there were no domestic cases of malaria and a competent vector (Anopheles minimus) was lacking in Taiwan but also by the evidence of no history of travel by the patient during the previous few years. Thus, because of the simplicity and feasibility of examining blood smears during routine differential diagnosis for patients suspected of having hemolytic anemia and living in rural areas, their use should be considered by physicians and clinical investigators. The titer of antibody reactivity against the Babesia parasite, in general, wanes during the course of chronic or subclinical infection. Previous studies (33, 34) indicate that an antibody titer of 1,024 or more indicates a relatively recent or acute infection, and a titer of 64 or less was suggested as being indicative of a chronic or subclinical infection. Indeed, a chronic or subclinical carrier state had been reported in animals infected with various species of Babesia and Theileria (3, 12, 27), and the persistence of elevated antibody titers was attributed to a subclinical or self-limited infection in humans (21, 28, 34). For the patient described here, the patient had a titer of 512 to the TW1 isolate and a titer of 64 to B. microti in June 1994, and a titer of 128 to TW1 accompanied by a lowgrade parasitemia was sustained for almost 3 months after routine treatment (Table 2). Although high titers of antibody may persist for several months in infected persons, an antibody response appears to wane within a year in treated individuals (21). Indeed, our results also indicate that the seroreactivity against the patient s Babesia parasites gradually fell to an undetectable level 6 months after retreatment. On the basis of these observations, we suggest that the present case of babesial infection may have been acquired a few months prior to the clinical investigations. The identity of a Babesia isolate can be verified by measuring seroreactivity with various sera obtained from Babesia-infected humans and animals. As described previously (7, 22, 29), the IFA test has a high degree of specificity for diagnosing chronic infection and identifying the species of the Babesia parasite. In general, the degree of cross-reactivity between known species of the Babesia parasite and the suspected agent of human babesiosis may eventually be used to identify the species responsible for human infection. In our study, the TW1 isolate had a strong cross-reactivity with antiserum against B. microti, but it had only weak or no reactivity with antiserum against canine Babesia. In addition, the ability of subinoculation of the patient s blood into hamsters to amplify the parasitemia may also facilitate the differential diagnosis of the Babesia isolate. On the basis of the morphological similarity and serological reactivity, the Babesia isolate in the present patient was identified as being closely related to the rodent parasite (B. microti). The prevalence of babesial infection among rural populations in Taiwan has not yet been surveyed. It is interesting that a fairly high ( 75%) prevalence of babesial infection had been reported in rodents on Taiwan (41), and these rodents were parasitized abundantly, possibly by the Ixodes tick vector. Thus, the risk of zoonotic transmission of babesial infection was assumed to be high among rural populations. Although the possibility of acquiring babesial infection was said to be rare among Taiwan aborigines (20), it is possible that a diagnostic serologic survey with babesial antigens derived from nonlocal isolates may partially account for the insensitivity of the tests. In addition, hamster inoculation of the suspected human blood, especially from a person with tick exposure, to amplify the prepatent parasitemia may also be required because of the relative scarcity of parasites during the subclinical or occult stages of infection. Thus, a serologic survey with antigens prepared from the babesial isolate of Taiwan would be required to verify the current status of babesial infection among rural populations in Taiwan. The transmission cycle of the etiologic agent (isolate TW1) needs to be identified. Further understanding of the risk of acquiring infection by rural populations will depend on the TABLE 2. Cross-reactivity of human (TW1) and rat isolates with sera from various Babesia-infected humans and animals Source and type of antiserum a Original antibody titer Reciprocal IFA titer b to: TW1 isolate Rat isolate c Human Anti-B. microti (GI strain) 1:2, Anti-B. microti (GI strain) 1:4, Anti-B. microti (Gray strain) 1:4, Canine Anti-B. canis 1:2, Anti-B. gibsoni 1:20, a Human and canine antisera were obtained from Sam R. Telford III and Patricia A. Conrad, respectively. b FITC-conjugated goat anti-human IgG and rabbit anti-dog IgG were used for analysis. c Babesia organism isolated from a rat near the patient s home.

5 454 SHIH ET AL. J. CLIN. MICROBIOL. identification of animal reservoirs and the tick vectors responsible for transmission. Although we have not yet identified the tick vector, the high prevalence of babesial infection in rodents provides evidence of the possibility of zoonotic transmission in Taiwan. Thus, improved serodiagnostic and molecular techniques (8, 27) may also be required to characterize Babesia species and elucidate the epidemiology of babesiosis in Taiwan. In conclusion, we reported the first laboratory-confirmed case of human babesiosis in Taiwan. The Babesia isolate (isolate TW1) was morphologically indistinguishable from and serologically related to the rodent parasite of B. microti. Our report also highlights the demand for increased vigilance for the detection of vector-borne infections that are not previously known to infect humans in Taiwan. Whether babesiosis is an emerging zoonosis in Taiwan remains to be determined. ACKNOWLEDGMENTS We thank Sam R. Telford III for providing anti-b. microti human sera and B. microti-infected hamster erythrocytes and Patricia A. Conrad for providing the dog serum with high titers of antibodies against B. gibsoni and B. canis. Ling-ling Lee (Department of Zoology, National Taiwan University) is also appreciated for the helpful pictorial guide for the species identification of the trapped rodents. This work was supported in part by a grant from the Department of Health (DOH85-TD-019) and Department of Defense (Medical Research 85). REFERENCES 1. Anderson, A. E., P. B. Cassaday, and G. R. Healy Babesiosis in man. Sixth documented case. Am. J. Clin. Pathol. 62: Benach, J. L., D. J. White, and J. P. McGovern Babesiosis in Long Island: host-parasite relationships of rodent- and human-derived Babesia microti isolates in hamsters. Am. J. Trop. Med. Hyg. 27: Bishop, R., B. Sohanpal, D. P. Kariuki, A. S. Young, V. Nene, H. Baylis, B. A. Allsopp, P. R. Spooner, T. T. Dolan, and S. P. 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